Galvanotaxis of human granulocytes: electric field jump studies

The static and dynamic responses of human granulocytes to an electric field were investigated. The trajectories of the cells were determined from digitized pictures (phase contrast). The basic results are: (i) The track velocity is a constant as shown by means of the velocity autocorrelation function. (ii) The chemokinetic signal transduction/response mechanism is described in analogy to enzyme kinetics. The model predicts a single gaussian for the track velocity distribution density as measured. (iii) The mean drift velocity induced by an electric field, is the product of the mean track velocity and the polar order parameter. (iv) The galvanotactic dose-response curve was determined and described by using a generating function. This function is linear in E for E < E ₀ = 0.78 V/mm with a galvanotaxis coefficient K _G of (–0.22 V/mm)^–1 at 2.5 mM Ca⁺⁺. For E > E ₀ the galvanotactic response is diminished. This inhibition is described by a second term in the generating function (–K _G · K _I (E – E ₀)) with an inhibition coefficient K _I of 3.5 (v) The characteristic time involved in directed movement is a function of the applied electric field strength: about 30 s at low field strengths and below 10 s at high field strengths. The characteristic time is 32.4 s if the cells have to make a large change in direction of movement even at large field strength (E jump). (vi) The lag-time between signal recognition and cellular response was 8.3 s. (vii) The galvanotactic response is Ca⁺⁺ dependent. The granulocytes move towards the anode at 2.5 mM Ca⁺⁺ towards the cathode at 0.1 mM Ca⁺⁺. (viii) The directed movement of granulocytes can be described by a proportional-integral controler. Offprint requests to: H. Gruler     

Studies on the morphology and taxonomy of three new myxosporeans of the genus Sinuolinea Davis, 1917 (Myxosporea: Sinuolineidae) infecting the urinary bladder of some marine fishes from the Shandong coast,China

The morphology and taxonomy of three new species of myxosporeans (Myxosporea: Sinuolineidae), Sinuolinea shandongensis n. sp., S. argyrosomi n. sp. and S. platycephali n. sp., parasitising marine fishes collected from the Yellow Sea off the coast of southeast Shandong, China, were investigated. These species, including spores and plasmodia, were found in the urinary bladder of their hosts. The diagnostic features of the new species are as follows. S. shandongensis n. sp. from Nibea albiflora: monosporous and disporous plasmodia, 29–37 × 29–27 m; spore inversely pyramidal or spherical, with smooth surface and fine, highly sinuous sutural line, 16.4±2.4 (15–20) × 16.1±1.1 (15–18) m; two spherical polar capsules located anteriorly and conspicuously separated from each other, 4.8±0.3 (4.5–5.5) m in diameter; length of polar filament 59–61 m; coelozoic. S. argyrosomi n. sp. from Argyrosomus argentatus: monosporous plasmodium, (33–42) × (24–25) m; spore subspherical to spherical, with smooth surface and highly sinuous sutural line, 18.1±1.0 (17–20) × 19.8±1.3 (18–22) m; two spherical polar capsules located anteriorly and conspicuously separated from each other, 4.4±0.2 (4–5) m in diameter; length of polar filament 47–50 m; coelozoic. S. platycephali n. sp. from Platycephalus indicus: monosporous, disporous and polyporous plasmodia, 25–42 × 17–31 m; spore spherical with smooth surface and highly sinuous sutural line, 14.5±0.5 (14–15) m in diameter; two spherical polar capsules located anteriorly and conspicuously separated from each other, 4.3±0.2 (4–4.5) m in diameter; coelozoic.     

Dendritic properties of uropod motoneurons and premotor nonspiking interneurons in the crayfish Procambarus clarkii Girard

Dendritic properties of uropod motoneurons and premotor nonspiking interneurons of crayfish have been studied using intradendritic recording and current injection. The input resistance of phasic motoneurons (5.20 ± 0.5 M; mean ± standard error) measured by injecting constant hyperpolarizing current was significantly lower than that of tonic motoneurons (10.3 ± 2.6 M; 0.02 < P < 0.05). The membrane time constant of phasic motoneurons (7.3 ± 0.9 ms) was also significantly shorter than that of tonic motoneurons (24.3 ± 2.5 ms; P < 0.001). Both types of motoneurons behaved linearly during hyperpolarization and sub-threshold depolarization. Nonspiking interneurons showed outward rectification upon depolarization. During hyperpolarization, their membrane behaved linearly and showed significantly higher input resistance (19.5 ± 2.5 M) than phasic and tonic motoneurons (P < 0.001). Their membrane time constant (38.0 ± 5.7 ms) was significantly longer than that of phasic motoneurons (P < 0.001) but not than that of tonic motoneurons (P > 0.05). In response to intracellular injection of sinusoidally oscillating current, phasic motoneurons showed one or two spikes per depolarization period irrespective of oscillating frequency ranging from 1 to 16 Hz. Tonic motoneurons showed larger numbers of spikes per stimulus period at lower frequencies. Nonspiking interneurons also showed phase-locked effects on the motoneuron spike activity. The effective frequency range over which injected oscillating current could modulate motoneuron spike activity was similar for tonic motoneurons and nonspiking interneurons.     

Davisia hexagrammi n. sp. (Myxosporea: Sinuolineidae) parasitic in the urinary bladder of marine fishes from the coasts of the Yellow Sea and Bohai Bay,China

A new species of myxosporean (Sinuolineidae), Davisia hexagrammi n. sp. from marine fishes collected from the coasts off Qingdao (Tsingtao) and Rongcheng on the Yellow Sea coast and Laizhou Bay, China, is described. Both spores and plasmodia were found in the urinary bladder of two hosts, Hexagrammos otakii Jordan & Starks and Agrammus agrammus (Temminck & Schlegel). The diagnostic features of D. hexagrammi n. sp. are: trophozoite monosporous or disporous; spore body spherical, 9.5-10.5 (9.9±0.5)× 10-12 (10.8±1.0) m in size and with a straight or slightly sinuous sutural line; two shell valves each with one long, hollow lateral appendage of 44-59 (52.4±6.9) m in length; two spherical polar capsules arranged anteriorly, well-separated from each other and 3.5-4 (3.8±0.2) m in diameter, coelozic; marine habitat.     

Thermoregulation in resting and active polar bears

Summary Polar bears (Ursus maritimus) regulate their body temperatures both physiologically and behaviourally proportional to their level of activity while within the thermoneutral zone. Core temperatures (T _c=36.9±0.5°C at rest) varied with the 4^th power of walking speed for the two subadult (220 kg) bears tested, whereas subcutaneous temperatures (T _sk=35.3±2.2°C at rest) were closely correlated withT _c but also varied with wind speed (v _a) and ambient temperature (T _a). Radiative fur temperatures (T _r) were closely correlated withT _a and negligibly withT _sk. Predictive equations for these temperature relationships were derived by regression analysis. Maximum rates of heat storage (S _max) were above that predicted from the literature implying that the polar bear is an energetically costly walker. Radiative heat losses of a resting polar bear amount to between 36–67% of the metabolism and assuming a respiratory heat loss of 7–10%, convective heat losses (by difference) would thus range from 33–64%. When walking, the cooling of the fur surface by forced convection and the pendulum effect of the moving legs of the bear lead to estimated convective heat losses on the order of 75% of the heat production while radiative losses are reduced to 13–22%. Increasing wind speeds enhance further this reciprocal effect.     

Characteristics of L-Type Ca2+ Channels in the Membrane of Mesenteric Artery Myocytes from Genetically Hypertensive Rats

Using the standard voltage-clamp technique in the whole-cell mode, we studied the characteristics of barium currents (I _Ba; Ba²⁺ concentration in the external solution was 5 mM) carried through L-type Ca²⁺ channels in the membrane of myocytes of the resistive mesenteric artery from normotensive and genetically hypertensive rats (NR and GHR, respectively). To perforate the membrane, we used amphotericin B. The arbitrary density of I _Ba through the plasma membrane of GHR myocytes significantly exceeded this parameter in the NR group. For both animal groups, activation curves plotted as the dependence of the membrane conductance (G _Ba) on the membrane potential were not significantly different: the membrane potential for half activation (V _0.5) of I _Ba in the NR myocytes was equal to 1.0 ± 0.3 mV with slope factor k = 6.3 ± 0.4 mV, whereas in the GHR myocytes V _0.5 = -1.6 ± 0.2 mV and k = 6.2 ± 0.5 mV. The stationary inactivation curves for I _Ba differed significantly: in the NR myocytes, V _0.5 = -24.2 ± 0.4 mV and k = 8.3 ± 0.2 mV, whereas in the GHR myocytes such parameters were, respectively, -21.4 ± 0.4 and 8.7 ± 0.3 mV. The pattern of intersection of stationary activation and stationary inactivation curves for I _Ba was indicative of the existence of a window current, i.e., the non-inactivating component of I _Ba within the -40 to ±20 mV range; the phenomenon was clearly pronounced in the GHR myocytes. Differences in the arbitrary density of integral I _Ba and window current were observed. These differences can cause an increased tone of the blood vessels in hypertensive animals.     

Fine-scale movement and assimilation of carbon in saltmarsh and mangrove habitat by resident animals

Despite theories of large-scale movement and assimilation of carbon in estuaries, recent evidence suggests that in some estuaries much more limited exchange occurs. We measured the fine-scale movement and assimilation of carbon by resident macroinvertebrates between adjacent saltmarsh and mangrove habitats in an Australian estuary using ¹³C analysis of animals at different distances into adjacent patches of habitat. ¹³C values of crabs (Parasesarma erythrodactyla –15.7 ± 0.1, Australoplax tridentata –14.7 ± 0.1) and slugs (Onchidina australis –16.2 ± 0.3) in saltmarsh closely matched that of the salt couch grass Sporobolus virginicus (–15.5 ± 0.1). In mangroves, ¹³C values of crabs (P. erythrodactyla –22.0 ± 0.2, A. tridentata –19.2 ± 0.3) and slugs (–19.7 ± 0.3) were enriched relative to those of mangroves (–27.9 ± 0.2) but were more similar to those of microphytobenthos (–23.7 ± 0.3). The ¹³C values of animals across the saltmarsh-mangrove interface fitted a sigmoidal curve, with a transition zone of rapidly changing values at the saltmarsh-mangrove boundary. The width of this transition indicated that the movement and assimilation of carbon is limited to between 5 and 7 m. The ¹³C values of crabs and slugs, especially those in saltmarsh habitat, clearly indicate that the movement and assimilation of carbon between adjacent saltmarsh and mangrove habitat is restricted to just a few metres, although some contribution from unmeasured sources elsewhere in the estuary is possible. Such evidence demonstrating the extent of carbon movement and assimilation by animals in estuarine habitats is useful in determining the spatial arrangement of habitats needed in marine protected areas to capture food web processes.     

The Soil Flagellate Heteromita globosa Accelerates Bacterial Degradation of Alkylbenzenes through Grazing and Acetate Excretion in Batch Culture

The impact of grazing by soil flagellates Heteromita globosa on aerobic biodegradation of benzene by Pseudomonas strain PS+ was examined in batch culture. Growth of H. globosa on these bacteria obeyed Monod kinetics (_max, 0.17 ± 0.03 h^–1; K_s, 1.1 ± 0.2 × 10⁷ bacteria mL^–1) and was optimal at a bacteria/ flagellate ratio of 2000. Carbon mass balance showed that 5.2% of total [ring-U-¹⁴C]benzene fed to bacteria was subsequently incorporated into flagellate biomass. Growth-inhibiting concentrations (IC₅₀) of alkylbenzenes (benzene, toluene, ethylbenzene) were inversely related with their octanol/ water partitioning coefficients, and benzene was least toxic for bacteria and flagellates with IC₅₀ values of 4392 (± 167) M and 2770 (± 653) M, respectively. The first-order rate constant for benzene degradation (k₁, 0.48 ± 0.12 day^–1) was unaffected by the presence or absence of flagellates in cultures. However, the rate of benzene degradation by individual bacteria averaged three times higher in the presence of flagellates (0.73 ± 0.13 fmol cell^–1 h^–1) than in their absence (0.26 ± 0.03 fmol cell^–1 h^–1). Benzene degradation also coincided with higher levels of dissolved oxygen and a higher rate of nitrate reduction in the presence of flagellates (p < 0.02). Grazing by flagellates may have increased the availability of dissolved oxygen to a smaller surviving population of bacteria engaged in the aerobic reactions initiating benzene degradation. In addition, flagellates may also have increased the rate of nitrate reduction through the excretion of acetate as an additional electron donor for these bacteria. Indeed, acetate was shown to progressively accumulate in cultures where flagellates grazed on heat-killed bacteria. This study provided evidence that grazing flagellates stimulate bacterial degradation of alkylbenzenes and provide a link for carbon cycling to consumers at higher trophic levels. This may have important implications for bioremediation processes.     

Differential effect of ethanol on muscarinic cholinergic binding to rat and locust neural membranes

We have compared the effect of ethanol, a membrane perturbant, on the muscarinic binding sites in neural membranes from a vertebrate (rat) and an insect (locust). The binding of the muscarinic antagonist [³H]quinuclidinyl benzilate ([³H]QNB) to both rat and locust neural membranes was inhibited by ethanol at 10–500 mM concentrations; but this inhibition was greater in the locust. Ethanol (500 mM) increased the apparent dissociation constant (K d) of [³H]QNB binding to rat membranes from 0.13±0.01 nM in control to 0.20±0.02 nM; there was also an small but significant reduction in the number of binding sitesB _max. In locust, 500 mM ethanol reduced theB _max of [³H]QNB binding from 590±30 in control to 320±40 pmol/g protein; no significant alteration in theK _D was detected. The dissociation rate constant (k _off) of [³H]QNB increased from 0.020±0.003 in controls to 0.031±0.004 (min^–1) in the presence of 500mM ethanol, the association rate constant (k _on) did not change significantly. In locust, 500 mM ethanol did not affect eitherk _on ork _off. Competition experiments revealed that the binding affinities of both the agonist carbamylcholine and the antagonist atropine to the rat membranes were reduced in the presence of ethanol. In contrast, ethanol caused no alteration in the binding affinities of these ligands to the locust membranes. This differential effect of ethanol on rat and locust muscarinic binding suggests a difference in the hydrophobic domains and/or the membrane interactions of the muscarinic receptors in the two species.     

Effects of the anion transport inhibitor,SITS, on the proximal straight tubule of the rabbit perfusedin vitro

Summary Conventional microelectrodes were used to study the effects of SITS (4-acetamido-4-isothiocyanostilbene-2,2-disulfonate) on the basolateral membrane potentialVbl of the superficial proximal straight tubule (PST) of the rabbit kidney perfusedin vitro. Addition of 0.1mm SITS to the bathing solution resulted in a slow and irreversible hyperpolarization ofVbl from –42.5±1.17 (37) mV to –77.3±0.83 (52) mV. The new steady-state potential was reached in 10 to 15 min and was accompanied by visible cell swelling. Associated with thisVbl hyperpolarization was: 1) an increased steady-state depolarization (from 6.2±0.77 (17) mV to 25.7±0.83 (29) mV) in response to increasing bath potassium concentration from 5 to 16.7mm (HK); 2) a decreased transient depolarization (from 19.8±1.88 (8) mV to 0.43±0.37 (8) mV) in response to decreasing bath bicarbonate concentration from 22 to 6.6mm at constant bath pH (L-HCO₃); and 3) inhibition of a depolarizing overshoot and a decreased steady-state depolarization (from 35.9±1.84 (12) mV to 4.7±1.37 (13) mV) in response to reducing bath sodium concentration from 144 to zero (0-Na). Sodium, chloride and NMDG (N-methyl-d-glucamine) were used as the substituting ions, respectively. These results are consistent with the presence of a coupled sodium-bicarbonate carrier in the basolateral membrane which is electrogenic and SITS inhibitable. Comparison of the time course of SITS effects on these ion-substitution responses suggests that the inhibition of the bicarbonate exit pathway(s) is the primary event and that the changes inVbl and in the steady-stateVbl responses to HK and 0-Na are secondary events which may be related to changes in intracellular composition and/or basolateral membrane properties.     

Water and salt permeability of gastric vesicles

Summary Volume-dependent changes in light scatter have been shown to be a linear function of the osmotic gradient imposed upon gastric vesicles purified from hog gastric mucosa. Observation of the light scattered 90° to incident, using the Durrum stop flow system D-110, indicates that the vesicles exposed to hypertonic medium undergo rapid shrinkage due to water loss from the vesicle interior. The rate constant for this water movement is 1.1±0.09 sec^–1 (n=10) and is linearly dependent on temperature between 16 and 36°C. The activation energy of 13.93±0.60 kcal mole^–1 (n=3), calculated from an Arrhenius plot, is inconsistent with water movement facilitated by a large-pore aqueous channel. A slower reswell phase, dependent on solute entry into the intravesicular space, follows the water-dependent shrink phase. KCl entry, studied because of the intravesicular requirement for active K⁺/H⁺ transport, exhibits two entry stages. The faster, described by a single exponential imposed upon a constantly sloping background, has a rate constant of 7.75±0.48×10^–3 sec^–1 (n=15). The slower phase, which typically accounts for 90% of the reswell process, demonstrates a rate constant of 1.94±0.23×10^–4 sec^–1 (n=15). In the presence of valinomycin or nigericin, two fast rate constants and one slow rate constant of swelling are observed. The rate constant of the faster reswell phase is increased from 7.75±0.48×10^–3 sec^–1 (n=15) to 15.74±3.7×10^–3 sec^–1 (n=5) and 17.23±3.4×10^–3 (n=3) by the addition of nigericin (1 g ml^–1) and valinomycin (4.5 m), respectively. The second part of the faster reswell phase is approximately that seen in the control population. Transport-dependent volume changes of significant magnitude can be demonstrated following the addition of ATP to vesicles equilibrated with 150mm KCl. The volume change is a function of HCl leak rate and is abolished by ionophores which eliminate the transport-dependent pH gradient. So ₄ ^–- substitution, which eliminates the overshoot phenomena observed in KCl medium, also eliminates the shrinkage resulting from ATP addition.     

Cold tolerance of Littorinidae from southern Africa: intertidal snails are not constrained to freeze tolerance

All intertidal gastropods for which cold tolerance strategies have been assessed have been shown to be freeze tolerant. Thus, freeze tolerance is considered an adaptation to the intertidal environment. We investigated the cold tolerance strategies of three species of subtropical and temperate snails (Gastropoda: Littorinidae) to determine whether this group is phylogenetically constrained to freeze tolerance. We exposed dry acclimated and wet rehydrated snails to low temperatures to determine temperature of crystallisation (Tc), lower lethal temperature and LT₅₀ and to examine the relationship between ice formation and mortality. Tc was lowest in dry Afrolittorina knysnaensis (–13.6±0.4 °C), followed by dry Echinolittorina natalensis (–10.9±0.2 °C) and wet A. knysnaensis (–10.2±0.2 °C). The Tc of both A. knysnaensis and E. natalensis increased with rehydration, whereas Tc of dry and wet Afrolittorina africana did not differ (–9.6±0.2 and –9.0±0.2 °C respectively). Wet snails of all species exhibited no or low survival of inoculative freezing, whereas dry individuals of A. knysnaensis could survive subzero temperatures above –8 °C when freezing was inoculated with ice. In the absence of external ice, Afrolittorina knysnaensis employs a freeze-avoidance strategy of cold tolerance, the first time this has been reported for an intertidal snail, indicating that there is no family-level phylogenetic constraint to freeze tolerance. Echinolittorina natalensis and A. africana both showed pre-freeze mortality and survival of some internal ice formation, but were not cold hardy in any strict sense.     

Excitation-calcium release uncoupling in aged single human skeletal muscle fibers

The biological mechanisms underlying decline in muscle power and fatigue with age are not completely understood. The contribution of alterations in the excitation-calcium release coupling in single muscle fibers was explored in this work. Single muscle fibers were voltage-clamped using the double Vaseline gap technique. The samples were obtained by needle biopsy of the vastus lateralis (quadriceps) from 9 young (25–35 years; 25.9 ± 9.1; 5 female and 4 male) and 11 old subjects (65–75 years; 70.5 ± 2.3; 6 f, 5 m). Data were obtained from 36 and 39 fibers from young and old subjects, respectively. Subjects included in this study had similar physical activity. Denervated and slow-twitch muscle fibers were excluded from this study. A significant reduction of maximum charge movement (Q_max) and DHP-sensitive Ca current were recorded in muscle fibers from the 65–75 group. Q_max values were 7.6 ± 0.9 and 3.2 ± 0.3 nC/F for young and old muscle fibers, respectively (P < 0.01). No evidences of charge inactivation or interconversion (charge 1 to charge 2) were found. The peak Ca current was (–)4.7 ± 0.08 and (–)2.15 ± 0.11 A/F for young and old fibers, respectively (P < 0.01). The peak calcium transient studied with mag-fura-2 (400 m) was 6.3 ± 0.4 m and 4.2 ± 0.3 m for young and old muscle fibers, respectively. Caffeine (0.5 mm) induced potentiation of the peak calcium transient in both groups. The decrease in the voltage-/ Ca-dependent Ca release ratio in old fibers (0.18 ± 0.02) compared to young fibers (0.47 ± 0.03) (P < 0.01), was recorded in the absence of sarcoplasmic reticulum calcium depletion. These data support a significant reduction of the amount of Ca available for triggering mechanical responses in aged skeletal muscle and, the reduction of Ca release is due to DHPR-ryanodine receptor uncoupling in fast-twitch fibers. These alterations can account, at least partially for the skeletal muscle function impairment associated with aging.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association, and National Institutes of Health (2-P60AG18484-06)     

Root and stem xylem embolism,stomatal conductance,and leaf turgor in Acer grandidentatum populations along a soil moisture gradient

The objective of this study was to determine how adjustment in stomatal conductance (g ^s) and turgor loss point (_tlp) between riparian (wet) and neighboring slope (dry) populations of Acer grandidentum Nutt. was associated with the susceptibility of root versus stem xylem to embolism. Over two summers of study (1993–1994), the slope site had substantially lower xylem pressures (_px) and g ^s than the riparian site, particularly during the drought year of 1994. The _tlp was also lower at the slope (-2.9±0.1 MPa; all errors 95% confidence limits) than at riparian sites (-1.9±0.2 MPa); but it did not drop in response to the 1994 drought. Stem xylem did not differ in vulnerability to embolism between sites. Although slope-site stems lost a greater percentage of hydraulic conductance to embolism than riparian stems during the 1994 drought (46±11% versus 27±3%), they still maintained a safety margin of at least 1.7 MPa between midday _px and the critical pressure triggering catastrophic xylem embolism (_pxCT). Root xylem was more susceptible to embolism than stem xylem, and there were significant differences between sites: riparian roots were completely cavitated at -1.75 MPa, compared with -2.75 MPa for slope roots. Vulnerability to embolism was related to pore sizes in intervessel pit membranes and bore no simple relationship to vessel diameter. Safety margins from _pxCT averaged less than 0.6 MPa in roots at both the riparian and slope sites. Minimal safety margins at the slope site during the drought of 1994 may have led to the almost complete closure of stomata (g _s=9±2 versus 79±15 mmol m^-2 s^-1 at riparian site) and made any further osmotic adjustment of _tlp non-adaptive. Embolism in roots was at least partially reversed after fall rains. Although catastrophic embolism in roots may limit the minimum for gas exchange, partial (and reversible) root embolism may be adaptive in limiting water use as soil water is exhausted.     

Binding of chloride and a disulfonic stilbene transport inhibitor to red cell band 3

Summary The effect of chloride on 4,4-dibenzamido-2,2-disulfonic stilbene (DBDS) binding to band 3 in unsealed red cell ghost membranes was studied in buffer [NaCl (0 to 500mm) + Na citrate] at constant ionic strength (160 or 600mm). pH 7.4, 25°C. In the presence of chloride, DBDS binds to a single class of sites on band 3. At 160mm ionic strength, the dissociation constant of DBDS increases linearly with chloride concentration in the range [Cl]=450mm. The observed rate of DBDS binding to ghost membranes, as measured by fluorescence stopped-flow kinetic experiments, increases with chloride concentration at both 160 and 600mm ionic strength. The equilibrium and kinetic results have been incorporated into the following model of the DBDS-band 3 interaction: The equilibrium and rate constants of the model at 600mm ionic strength areK ₁=0.67±0.16 m,k ₂=1.6±0.7 sec^–1,k _–2=0.17±0.09 sec^–1,K ₁=6.3±1.7 m,k ₂=9±4 sec^–1 andk _–2=7±3 sec^–1. The apparent dissociation constants of chloride from band 3,K _Cl, are 40±4mm (160mm ionic strength) and 11±3mm (600mm ionic strength). Our results indicate that chloride and DBDS have distinct, interacting binding sites on band 3.     

Progressively developed myocardial apoptotic cell death during late phase of reperfusion

Myocardial apoptosis is primarily triggered during reperfusion (R). The aim of this study was to test the hypothesis that R-induced apoptosis develops progressively during the late phase of R, and that R-induced apoptosis is associated with changes in expression of anti- and pro-apoptotic proteins and infiltrated inflammatory cells. Thirty-one dogs were subjected to 60 min of left anterior descending coronary occlusion followed by 6, 24, 48, and 72 h R, respectively. There was no group difference in collateral blood flow, measured by colored microspheres during ischemia. Necrotic cell death (TTC staining) was significantly increased during R, starting at 27 ± 2% at 6 h R and increasing to 41 ± 2% at 24 h R. There was no further change at 48 (37 ± 3%) and 72 (36 ± 6%) h R, respectively. TUNEL positive cells (% total normal nuclei) in the peri-necrotic zone progressively increased from 6 (26 ± 2^*) to 24 (38 ± 1^*), 48 (48 ± 3^*) and 72 (59 ± 4^*) h R, respectively. The number of detected TUNEL positive cells at these time points was consistent with an increased intensity of DNA ladders, identified by agarose gel electrophoresis. Compared with normal tissue, western blot analysis showed persistent reduction in expression of anti-apoptotic protein Bcl-2 from 6 (16 ± 0.8%^*) to 72 h R (78 ± 2%^*), and increase in expression of pro-apoptotic proteins including Bax from 6 (30 ± 3%^*) to 72 h R (66 ± 3%^*), and p53 from 6 (12 ± 1%^*) to 72 h R (91 ± 2%^*), respectively. Immunohistochemical staining revealed that infiltrated neutrophils (mm² myocardium) were significantly correlated with development of necrotic and apoptotic cell death from 6 to 24 h R, respectively (P < 0.05), while large macrophage infiltration seen during 48 to 72 h R were correlated with apoptotic cell death (P < 0.05). These results indicate that 1) necrosis peaked at 24 h R when apoptosis was still progressively developing during later R; 2) changes in Bcl-2 family and p53 proteins may participate in R-induced myocardial apoptosis; 3) inflammatory cells may play a role in triggering cell death during R. ^* P < 0.05 vs. normal nuclei and tissue; P < 0.01 vs. 6 h R.     

Influence of five pyrethroid insecticides on microbial populations and activities in soil

Laboratory tests were conducted to determine the effects of five pyrethroid insecticides—permethrin (FMC 33297) [3-phenoxybenzyl (±)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate]; FMC 45498 [(S)--cyano-3-phenoxybenzyl-(R)-cis-2-(2,2-dibromovinyl)-3,3-dimethylcyclopropanecarboxylate]; Shell WL 41706 [(±)--cyano-3-phenoxybenzyl 2,2,3,3-tetramethylcyclopropane-carboxylate]; Shell WL 43467 [(±)--cyano-3-phenoxy benzyl (±)-cis,trans-2-(2,2-dichlorovinyl)-3,3-dimethylcyclopropanecarboxylate]; and Shell WL 43775 [(±)--cyano-3-phenoxybenzyl (±)-2-(4-chlorophenyl)-3-methylbutyrate]—at 0.5 and 5g/g on microbial populations and activities in a sandy loam. The insecticides had antimicrobial activity in early stages of incubation. The populations recovered after 2 to 4 weeks and stimulatory effects on populations were also observed in later stages. No inhibition of acetylene (C₂H₂) reduction was evident with any of the insecticides. However, WL 43467 at both concentrations and permethrin and WL 41706 at 5 g/g increased nitrification after 4 weeks. Soil microbial respiration, as indicated by oxygen consumption, increased with increasing concentration of insecticides, suggesting the possibility of microbial degradation of the insecticides. Dehydrogenase activity showed that none of the insecticides inhibited formazan (2,3,5-triphenyltetrazolium formazan) formation, whereas urease activity was stimulated in most cases. The studies indicated that some of the pyrethroid insecticides may exert transient effects on populations and activities of the microflora in a sandy loam, but these were short-lived and minor in nature.     

pH i -Dependent membrane conductance of proximal tubule cells in culture (OK): Differential effects on K+- and Na+-conductive channels

Summary Confluent monolayers of the established opossum kidney cell line were exposed to NH₄Cl pulses (20 mmol/liter) during continuous intracellular measurements of pH, membrane potential (PD _m ) and membrane resistance (R _m) in bicarbonate-free Ringer. The removal of extracellular NH₄Cl leads to an intracellular acidification from a control value of 7.33±0.08 to 6.47±0.03 (n=7). This inhibits the absolute K conductance (g _K+), reflected by a decrease of K⁺ transference number from 71±3% (n=28) to 26±6% (n=5), a 2.6±0.2-fold rise ofR _m, and a depolarization by 24.2±1.5 mV (n=52). In contrast, intracellular acidification during a block ofg _K+ by 3 mmol/liter BaCl₂ enhances the total membrane conductance, being shown byR _m decrease to 68±7% of control and cell membrane depolarization by 9.8±2.8 mV (n=17). Conversely, intracellular alkalinization under barium elevatesR _m and hyperpolarizes PD _m . The replacement of extracellular sodium by choline in the presence of BaCl₂ significantly hyperpolarizes PD _m and increasesR _m, indicating the presence of a sodium conductance. This conductance is not inhibited by 10^–4 mol/liter amiloride (n=7). Patch-clamp studies at the apical membrane (excised inside-out configuration) revealed two Na⁺-conductive channels with 18.8±1.4 pS (n=10) and 146 pS single-channel conductance. Both channels are inwardly rectifying and highly selective towards Cl^–. The low-conductive channel is 4.8 times more permeable for Na⁺ than for K⁺. Its open probability rises at depolarizing potentials and is dependent on the pH of the membrane inside (higher at pH 6.5 than at pH 7.8).     

Effect of osmotic gradient on ADH-induced intramembranous particle aggregates in toad bladder

Summary Paired toad urinary bladders were prepared without or with an osmotic gradient (175 mosm) across them, stimulated for 2.5 (n=6), 5 (n=6), 30 (n=6) or 60 (n=6) min with ADH (20 mU/ml), and studied by freeze-fracture electron microscopy. Water permeability at these times was assessed in additional bladders (n=6 for each case) after tissue fixation according to the technique of Eggena. After both 60 and 30 min of ADH stimulation, the presence of a gradient compared with the absence of one was associated with fewer aggregates (242±35vs. 382±14 ×235 m^–2 at 60 min,P<0.01; 279±36vs. 470±51 ×235 m^–2 at 30 min,P<0.01) and lower water permeability (8.4±1.1vs. 18.8±1.8g×min^–1×cm^–1 ×mosm ^–1 at60min,P<0.005; 9.2±1.0vs. 22.0±2.1 g ×min^–1×cm^–2×mosm ^–1 at 30 min,P<0.001). In addition, with a gradient both maximum water permeability and maximum aggregate frequency were reached nearly together; a similar correspondence occurred without a gradient. We conclude that in the presence of an osmotic gradient both the ADH-associated aggregates and the water permeability response to ADH are prevented from reaching the higher levels observed in bladders not exposed to a gradient.     

Differences in protein patterns of gill epithelial cells of the fish Gillichthys mirabilis after osmotic and thermal acclimation

Different protein patterns in gill epithelium of a euryhaline and eurythermal teleost fish (Gillichthys mirabilis, Family Gobiidae) in response to long-term (2 months) osmotic and thermal acclimation were found for the first time. Gill epithelial cells were isolated to remove extracellular proteins and quantify specialized cell types. Chloride cells were identified on the basis of size (>10 m) and bright appearance after [2-(p-dimethylaminostyryl)-1-methyl-pyridinium-iodine] staining. Small mitochondria-rich cells were <5 m in diameter and showed intermediate fluorescence. Abundance of chloride cells and small mitochondria-rich cells was significantly influenced by osmotic but not thermal acclimation (dilute seawater/25°C: 1.4±0.2% chloride cells, 11.9±4.6% small mitochondria-rich cells; seawater/25°C: 2.4±0.6% chloride cells, 2.2±1.3% small mitochondria-rich cells; seawater/10°C: 2.9±0.3% chloride cells, 1.2±0.7% small mitochondria-rich cells). Pavement cells, identified by low fluorescence and intermediate size (5–10 m), largely predominated under all conditions (>85% of cells). Thus, they represented the major protein source in gill epithelium. Differences in protein patterns were detectable using two-dimensional but not one-dimensional electrophoresis. Of 602 proteins identified by charge and molecular weight properties, only two were induced by high temperature (25°C) and three in response to cold acclimation (10°C). Nine proteins were induced in diluted seawater-acclimated fish, whereas no seawater-induced proteins were found. We hypothesize that proteins induced under dilute seawater conditions are important for the function of pavement cells in gills of hyper-osmoregulating G. mirabilis.Abbreviations BCA bicinchoninic acid - BSS balanced salt solution - CC chloride cells - CLB cell lysis buffer - DASPMI [2-(p-dimethylaminostryryl)-1-methylpyridinium-iodine] - DSW diluted sea water - DTT dithiothreitol - EDTA ethylene-diaminetetraacetate - FW fresh water - IEF isoelectric focusing - PC pavement cells - PDA diacrylpiperazine - pI isoelectric point(s) - PMSF phenylmethanesulphonylfluoride - SDS sodium dodecyl sulfate - SMRC small mitochondria-rich cells - SW sea water - TEMED tetramethylenediamine